Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin

Consumer demand for fresh-like products with little or no degradation of nutritional and organoleptic properties has led to the study of new technologies in food preservation. Pulsed electric fields (PEF) is a nonthermal preservation method used to inactivate microorganisms mainly in liquid foods. Microorganisms in the presence of PEF suffer cell membrane damage. Nisin is a natural antimicrobial known to disrupt cell membrane integrity. Thus the combination of PEF and nisin represents a hurdle for the survival of Listeria innocua in liquid whole egg (LWE). L. innocua suspended in LWE was subjected to two different treatments: PEF and PEF followed by exposure to nisin. The selected frequency and pulse duration for PEF was 3.5 Hz and 2 micros, respectively. Electric field intensities of 30, 40 and 50 kV/cm were used. The number of pulses applied to the LWE was 10.6, 21.3 and 32. The highest extent of microbial inactivation with PEF was 3.5 log cycles (U) for an electric field intensity of 50 kV/cm and 32 pulses. Treatment of LWE by PEF was conducted at low temperatures, 36 degrees C being the highest. Exposure of L. innocua to nisin following the PEF treatment exhibited an additive effect on the inactivation of the microorganism. Moreover, a synergistic effect was observed as the electric field intensity, number of pulses and nisin concentration increased. L. innocua exposed to 10 IU nisin/ml after PEF exhibited a decrease in population of 4.1 U for an electric field intensity of 50 kV/cm and 32 pulses. Exposure of L. innocua to 100 IU nisin/ml following PEF resulted in 5.5 U for an electric field intensity of 50 kV/cm and 32 pulses. The model developed for the inactivation of L. innocua by PEF and followed by exposure to nisin proved to be accurate (p = 0.05) when used to model the inactivation of the microorganism by PEF in LWE with 1.2 or 37 IU nisin/ml. The presence of 37 IU nisin/ml in LWE during the PEF treatment for an electric field intensity of 50 kV/cm and 32 pulses resulted in a decrease in the population of L. innocua of 4.4 U.     

Inactivation of Salmonella spp. in liquid whole egg using pulsed electric fields, heat, and additives

This paper evaluates the lethal effectiveness on 7 different Salmonella serovars of the application, in static and continuous conditions, of pulsed electric fields (PEF) followed by heat treatments in liquid whole egg (LWE) with additives (EDTA or triethyl citrate-TC-). Compared to heat treatments, the PEF (25 kV/cm and 75-100 kJ/kg) followed by heat (52°C/3.5', 55°C/2', or 60°C/1') in LWE with 2% TC permitted the reduction of heat treatment time from 92 fold at 52°C to 3.4 fold at 60°C, and 4.8 fold at 52°C in LWE with EDTA for a 9-Log(10) reduction of the population of Salmonella Enteritidis. The new designed treatments inactivated more than 5 Log(10) cycles of Salmonella serovars Dublin, Enteritidis 4300, Enteritidis 4396, Typhimurium, Typhi, Senftenberg, and Virchow, both in static and continuous conditions. Conversely, current heat pasteurization treatments of 60°C/3.5' and 64°C/2.5' reduced 5 Log(10) cycles of various serovars of Salmonella but only 2 and 3-4 Log(10) cycles of Salmonella Senftenberg and Salmonella Enteritidis 4396, respectively. Soluble protein content (SPC) decreased 1.8%, 1.3%, and 5.0% after the successive application of PEF followed by heat at 52, 55, and 60°C in the presence of 2% TC, respectively, whereas 1.6% and 9.4% of SPC were reduced after heat pasteurization at 60 and 64°C, respectively. Results indicate that designed treatments could be an alternative to current heat pasteurization of LWE as showed higher lethal effectiveness against Salmonella serovars with a similar or even lower decrement of the soluble protein content.     

Evaluation of pulsed electric fields technology for liquid whole egg pasteurization

This investigation evaluated the lethal efficiency of pulsed electric fields (PEFs) to pasteurize liquid whole egg (LWE). To achieve this aim, we describe the inactivation of Salmonella Enteritidis and the heat resistant Salmonella Senftenberg 775 W in terms of treatment time and specific energy at electric field strengths ranging from 20 to 45 kV/cm. Based on our results, the target microorganism for this technology in LWE varied with intensity of the PEF treatment. For electric field strengths greater than 25 kV/cm, Salmonella Enteritidis was the most PEF-resistant strain. For this Salmonella serovar the level of inactivation depended only on the specific energy applied: i.e., 106, 272, and 472 kJ/kg for 1, 2, and 3 Log₁₀ reductions, respectively. The developed mathematical equations based on the Weibull distribution permit estimations of maximum inactivation level of 1.9 Log₁₀ cycles of the target Salmonella serovar in the best-case scenario: 250 kJ/kg and 25 kV/cm. This level of inactivation indicates that PEF technology by itself cannot guarantee the security of LWE based on USDA and European regulations. The occurrence of cell damage due to PEF in the Salmonella population opens the possibility of designing combined processes enabling increased microbial lethality in LWE.     

Effects of pH and temperature on inactivation of Salmonella typhimurium DT104 in liquid whole egg by pulsed electric fields

Pulsed electric field (PEF) exposes a fluid or semi-fluid product to short pulses of high-energy electricity, which can inactivate microorganisms. The efficacy of PEF treatment for pasteurisation of liquid eggs may be a function of processing temperature. In this study, effects of PEF, temperature, pH and PEF with mild heat (PEF + heat) on the inactivation of Salmonella typhimurium DT104 cells in liquid whole egg (LWE) were investigated. Cells of S. typhimurium were inoculated into LWE pH adjusted to 6.6, 7.2 or 8.2 at 15, 25, 30 and 40 °C. The PEF field strength, pulse duration and total treatment time were 25 kV cm⁻¹, 2.1 μs and 250 μs respectively. Cells of S. typhimurium in LWE at pH 7.2 were reduced by 2.1 logs at 40 °C and 1.8 logs at 30 °C. The PEF inactivation of S. typhimurium cells at 15 or 25 °C was pH dependent. Heat treatment at 55 °C for 3.5 min or PEF treatment at 20 °C resulted in c. 1-log reduction of S. typhimurium cells. Combination of PEF + 55 °C achieved 3-log reduction of S. typhimurium cells and was comparable to the inactivation by the heat treatment at 60 °C for 3.5 min. With further development, PEF + heat treatment may have an advantage over high-temperature treatment for pasteurisation of liquid eggs.     

Physical and structural changes in liquid whole egg treated with high-intensity pulsed electric fields

Liquid whole egg (LWE) is currently pasteurized through the application of heat; however, this treatment entails deleterious effects against some of the functional and technological properties of the product. In this study, the effect of high-intensity pulsed electric fields (HIPEF) processing (field strength: 19, 32, and 37 kV/cm) was compared to the traditional heat pasteurization (66 °C for 4.5 min). Different physical and structural characteristics of LWE, subjected or not to homogenization, were evaluated and compared, having the untreated LWE as a reference. Thermal treatment caused an increase in the viscosity of LWE, especially in nonhomogenized samples. HIPEF treatments did not modify the original color of LWE, whereas thermally treated samples developed an opaque appearance. LWE treated at 19 and 32 kV/cm exhibited a similar foaming capacity as fresh untreated egg, whereas thermal processing and PEF treatments of 37 kV/cm caused a substantial decrease in the foaming capacity of untreated liquid egg. Regarding the microstructure, the lipoprotein matrix appeared to be less affected by the HIPEF than by heat treatment if compared to the control. In addition, heat pasteurization had a significant impact on both the water-soluble protein content of the LWE samples (19.5% to 23.6% decrease) and the mechanical properties of the egg gels (up to 21.3% and 14.5% increase in hardness and cohesiveness, respectively). On the other hand, these parameters were not substantially affected in the HIPEF-treated samples. Heat-induced gels obtained from HIPEF-treated samples did not exhibit remarkable differences in the water-holding capacity (WHC) with respect to heat-pasteurized samples. PRACTICAL APPLICATION: The impact of high-intensity pulsed electric fields (HIPEF) processing on technological properties of liquid-whole egg was investigated and compared to that of thermal processing. Heat treatments cause a severe impact on the foaming capacity, the water-soluble protein content, and the rheological properties of liquid egg samples, whereas HIPEF treatments better preserved the food matrix structure. Microscopic observations support these results, thus suggesting that HIPEF-processing has potential application for the preservation of liquid egg through nonthermal means.     

Inactivation of Salmonella Typhimurium in orange juice containing antimicrobial agents by pulsed electric field

Combinations of different hurdles, including moderately high temperatures (<60 degrees C), antimicrobial compounds, and pulsed electric field (PEF) treatment, to reduce Salmonella in pasteurized and freshly squeezed orange juices (with and without pulp) were explored. Populations of Salmonella Typhimurium were found to decrease with an increase in pulse number and treatment temperature. At a field strength of 90 kV/cm, a pulse number of 20, and a temperature of 45 degrees C, PEF treatment did not have a notable effect on cell viability or injury. At and above 46 degrees C, however, cell death and injury were greatly increased. Salmonella numbers were reduced by 5.9 log cycles in freshly squeezed orange juice (without pulp) treated at 90 kV/cm, 50 pulses, and 55 degrees C. When PEF treatment was carried out in the presence of nisin (100 U/ml of orange juice), lysozyme (2,400 U/ml), or a mixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cell viability loss was increased by an additional 0.04 to 2.75 log cycles. The combination of nisin and lysozyme had a more pronounced bactericidal effect than did either nisin or lysozyme alone. An additional Salmonella count reduction of at least 1.37 log cycles was achieved when the two antimicrobial agents were used in combination. No significant difference (P > 0.05) in cell death was attained by lowering the pH value; only cell injury increased. Inactivation by PEF was significantly more extensive (P < 0.05) in pasteurized orange juice than in freshly squeezed orange juice under the same treatment conditions. This increase might be due to the effect of the chemical composition of the juices.     

Inactivation of Salmonella serovars in liquid whole egg by heat following irradiation treatments

Salmonella is a frequent contaminant on eggs and is responsible for foodborne illnesses in humans. Ionizing radiation and thermal processing can be used to inactivate Salmonella in liquid whole egg, but when restricted to doses that do not affect egg quality, these technologies are only partially effective in reducing Salmonella populations. In this study, the effect of ionizing radiation in combination with thermal treatment on the survival of Salmonella serovars was investigated. Of the six Salmonella serovars tested, Salmonella Senftenberg was the most resistant to radiation (Dgamma = 0.65 kGy) and heat (D(55 degrees C) = 11.31 min, z = 4.9 degrees C). Irradiation followed by thermal treatment at 55 or 57 degrees C improved the pasteurization process. Radiation doses as low as 0.1 kGy prior to thermal treatments synergistically reduced the D(55 degrees C) and D(57 degrees C) of Salmonella Senftenberg 3.6- and 2.5-fold, respectively. The D(55 degrees C) and D(57 degrees C) of Salmonella Typhimurium were reduced 2- and 1.4-fold and those of Salmonella Enteritidis were reduced 2- and 1.6-fold, respectively. Irradiation prior to thermal treatment would enable the reduction of heat treatment times by 86 and 30% at 55 and 57 degrees C, respectively, and would inactivate 9 log units of Salmonella serovars.     

Inactivation of Staphylococcus aureus by pulsed UV-light sterilization

Pulsed UV light is a novel technology to inactivate pathogenic and spoilage microorganisms in a short time. The efficacy of pulsed UV light (5.6 J/cm2 per pulse) for the inactivation of Staphylococcus aureus as suspended or agar seeded cells was investigated. A 12-, 24-, or 48-ml cell suspension in buffer was treated under pulsed UV light for up to 30 s, and 0.1 ml of sample was surface plated on Baird-Parker agar and incubated at 37 degrees C for 24 h to determine log reductions. Also, 0.1 ml of cell suspension in peptone water was surface plated on Baird-Parker agar plates, and the plates were treated under pulsed UV light for up to 30 s. The treated and untreated plates were incubated in the conditions described above. A 7- to 8-log CFU/ml reduction was observed for suspended and agar-seeded cells treated for 5 s or longer. In the case of suspended cells, the sample depth, time, treatment, and interaction were significant (P < 0.05). In the case of agar-seeded cells, the treatment time was significant (P < 0.05). Our results clearly indicate that pulsed UV technology has potential for the inactivation of pathogenic microorganisms.     

Inactivation of soybean lipoxygenase in soymilk by pulsed electric fields

The inactivation of soybean lipoxygenase by pulsed electric fields (PEF) was studied. Effects of PEF parameters (treatment time, pulse strength, pulse frequency and pulse width) were evaluated. Soymilk was exposed to pulsed strengths from 20 to 42 kV/cm for up to 1036 μs treatment time in square wave pulse of bipolar mode. Moreover, pulse frequency (100–600 Hz) and pulse width (1–5 μs) was also tested at constant pulsed treatment time of 345 μs and strength of 30 kV/cm. Residual activity of soybean lipoxygenase decreased with the increase of treatment time, pulse strength, pulse frequency and pulse width. The maximum inactivation of soybean lipoxygenase by PEF achieved 88% at 42 kV/cm for 1036 μs with 400 Hz of pulse frequency and 2 μs of pulse width at 25 °C. Inactivation of soybean lipoxygenase by pulsed electric fields was modeled using several kinetic models. Weibull distribution function was most suitable model describing the inactivation of soybean LOX as a function of pulsed electric fields process parameters. Moreover, reduction of soybean LOX activity related to the electric field strength could be well described by the Fermi model.     

Component release after exposure of Staphylococcus aureus cells to pulsed electric fields

The objective of this work was to get further insights on the mechanism of inactivation of bacterial cells by pulsed electric fields (PEF) through the study of the release of intracellular components after exposing Staphylococcus aureus cells in McIvlaine buffer (pH 7.0, 2 mS/cm) to PEF treatments of different intensity (18 and 25 kV/cm) and treatment times (from 20 to 400 μs). Release of most compounds, except proteins, was almost immediate after the treatment, but the relative amount released depended on the molecule studied. A good correlation between the release of the smallest components studied (particularly ions) and membrane permeabilization (as measured by NaCl sensitization and PI entry) was observed. On the other hand, results obtained suggested that S. aureus inactivation by PEF would be related to the exit of cytoplasmic proteins of a molecular weight higher than 6 kDa. Results obtained in this work indicated that increasing PEF treatment time would reduce the capability of S. aureus cells to repair the electropores formed and suggested that this might be due to the formation of pores of a larger size, which S. aureus cells would be unable to reseal in a situation of homeostasis loss.Industrial relevanceResults reported here can help to design more effective treatments for microbial inactivation using PEF on food, and therefore facilitate its industrial implementation.     

Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin

High-intensity pulsed electric fields (HIPEF) can be used as a nonthermal preservation method that is believed to enhance the effect of nisin on microorganisms such as Staphylococcus aureus. The survival of S. aureus inoculated into skim milk and treated with nisin, with HIPEF, or with a combination of nisin-HIPEF was evaluated. Nisin dose, milk pH, and HIPEF treatment time were the controlled variables that were set up at 20 to 150 ppm, pH 5.0 to 6.8, and 240 to 2,400 micros, respectively. HIPEF strength and pulse width were kept constant at 35 kV/cm and 4 micros, respectively. No reduction in S. aureus concentration was observed in skim milk at its natural pH after treatment with nisin, but 1.1 log units were recovered after 90 min of treatment at pH 5.0 with 150 ppm nisin. A reduction in viable S. aureus counts of 0.3 and 1.0 log unit in skim milk treated with HIPEF at its natural pH was observed at 240 and 2,400 micros, respectively. The nisin-HIPEF treatment design was based on a response surface methodology. The combined effect of nisin and HIPEF was clearly synergistic. However, synergism depended on pH. A maximum microbial inactivation of 6.0 log units was observed at pH 6.8, 20 ppm nisin, and 2,400 micros of HIPEF treatment time, whereas a reduction of over 4.5 log units was achieved when pH, nisin concentration, and HIPEF treatment times were set at 5.0, 150 ppm, and 240 micros, respectively.     

Inactivation of Yersinia enterocolitica by pulsed electric fields

The influence of growth conditions, treatment medium characteristics and PEF process parameters on the lethal effect on Yersinia enterocolitica of pulsed electric fields (PEF) treatments in batch has been investigated. Growth phase, temperature of growth, pH, conductivity of the treatment medium, pulse width and frequency of pulses did not influence the sensitivity of Y. enterocolitica to PEF. However, an A_w decrease from >0.99 to 0.93 of the treatment medium increased the PEF resistance of Y. enterocolitica with 3.5 log₁₀ cycles after a treatment of 22 kV/cm, 800 μs and 880 kJ/kg. Inactivation of Y. enterocolitica increased with the field strength, treatment time and total specific energy up to a maximum of 6 log₁₀ cycles after 28 kV/cm, 2000 μs and 3559 kJ/kg. A nonlinear relationship was found among the survival fraction and the treatment time or the specific energy that was accurately described by a mathematical model based on the Weibull distribution. The inactivation of Y. enterocolitica by PEF was characterized by maximum field strength thresholds. Above these thresholds, specific energy necessary to obtain a given level of inactivation scarcely decreased by increasing the electric field strength, and inactivation of Y. enterocolitica only depended on the specific energy applied.     

Predicting inactivation of Salmonella senftenberg by pulsed electric fields

Pulsed electric field inactivation of Salmonella senftenberg suspended in McIlvaine buffer of pH 7 and conductivity 2 mS/cm was investigated. In this study, square wave waveform pulses were used. After the same treatment time, inactivation of S. senftenberg depended neither on pulse width (1–15 μs) nor frequency of treatment (1–5 Hz). Survivor curves of S. senftenberg at different electric field strengths did not follow first-order kinetics. These survival curves were described by the log-logistic model proposed by Cole et al. [Cole, M. B., Davies, K. W., Munro, G., Holyoak, C. D., and Kilsby, D. C. (1993). A vitalistic model to describe the thermal inactivation of Listeria monocytogenes. Journal of Industrial Microbiology, 12, 232–239]. Comparison of measured and estimated values showed that this model accurately described the inactivation of S. senftenberg by high electric field pulses in the range of 12–28 kV/cm.     

Membrane fatty acids composition and fluidity modification in Salmonella Typhimurium by culture temperature and resistance under pulsed electric fields

Effects of various growth temperatures on cell membrane fatty acid composition of Salmonella Typhimurium (S. Typhimurium) and the resistance to Pulsed Electric Fields (PEF) treatments, as well as PEF combining with mild-thermal (35, 45, 55 °C) treatments were investigated. Results indicated that the PEF resistance of S. Typhimurium at stationary phase was varied markedly at different growth temperatures. S. Typhimurium grown at 45 °C exhibited greater PEF resistance than cells grown at a lower temperature. Alteration of membrane fatty acid composition decreased in the unsaturated to saturated fatty acids ratio (UFA/SFA) and increased in cyclopropane fatty acids (CFA) proportion as growth temperature increased. It was found that the PEF resistance of S. Typhimurium at stationary phase was in a membrane fluidity dependent manner. Thermal combined PEF treatment improved the PEF lethality which indicated that the PEF resistance of S. Typhimurium was greatly affected by the fluidity of cytomembrane.     

Inactivation of papain by pulsed electric fields in a continuous system

Papain (E.C. 3.4.22.2), a cysteine protease in papaya, was activated with reducing agents and treated with pulsed electric fields (PEF) in a continuous system at 10°C. Irreversible reduction of activity was observed in PEF-treated papain after 24 h storage at 4°C. Oxidation of papain active site, a cyteine residue, was not the major cause of papain inactivation by PEF. Temperature did not increase over 35°C during PEF treatment. The pH of papain solution was not changed after PEF treatment. Structural change was observed in PEF-treated papain by CD analysis. Inactivation of PEF-treated papain was related to the loss of α-helix structure. Heating at 60-80°C for 2 min did not significantly reduce the activity of papain.     

Inactivation ofEscherichia coliinoculated in liquid whole egg by high hydrostatic pressure

The resistance ofEscherichia coliin liquid whole egg was studied at several pressures (300, 350, 400 and 450MPa), temperatures (50, 20, 2 and –15°C) and times (5, 5+5, 10, 5+5+5, 15min). The highest reduction was obtained at 50°C (about 7log₈units). At 20 and –15°CE. coliwas more resistant to pressure than at 50 and 2°C. The intermittent treatments were more effective than continuous treatments at lower pressures (350MPa). The destruction increases upon increasing the pressure and the time treatment. Survivor curves were studied at 400MPa for two temperatures (20 and 2°C) and different times (0–60min), obtaining a decimal reduction time of 14.1min at 20°C and 9.5min at 2°X.     

Inactivation of Saccharomyces cerevisiae and Bacillus cereus by pulsed electric fields technology

The effect of pulsed electric field (PEF) treatment, applied in a continuous system, on Saccharomyces cerevisiae and Bacillus cereus cells and spores was investigated. S. cerevisiae inoculated into sterilised apple juice and B. cereus cells and spores inoculated into sterilised 0.15% NaCl were treated with electric field strengths of 10–28 kV/cm using an 8.3 pulse number and with pulse numbers of 4.2–10.4 at 20 kV/cm, respectively. The inactivation of S. cerevisiae depended on the electric field intensity and number of pulses. The yeast inactivation increased when the applied electric field intensity and pulse number were increased. Approximately four log cycles reduction was achieved in apple juice using 10.4 pulses at 20 kV/cm. B. cereus cells were less sensitive to PEF treatment. The reduction in microbial count of B. cereus cells was hardly more than one log cycle using 10.4 pulses at 20 kV/cm. The applied PEF treatment was ineffective on Bacillus cereus spores.     

Inactivation of Escherichia coli O157:H7 in liquid whole egg using combined pulsed electric field and thermal treatments

Inactivation of Escherichia coli O157:H7 in liquid whole egg using thermal and pulsed electric field (PEF) batch treatments, alone and in combination with each other, was investigated. Electric field intensities in the range from 9 to 15 kV/cm were used in the study. The threshold temperature for thermal inactivation alone was 50 °C. PEF enhanced the inactivation of E. coli O157:H7 when the sample temperature was higher than the thermal threshold temperature. The maximum inactivation of E. coli O157:H7 obtained using thermal treatment alone was ∼2 logs at 60 °C. However, combined heat and PEF treatments resulted in up to 4 log reduction of the pathogen. The kinetic rate constants k_TE for combined treatments at 55 °C varied from 0.025 to 0.119 pulse⁻¹ whereas the rate constants at 60 °C ranged from 0.034 to 0.228 pulse⁻¹. These results indicated a synergy between temperature and electric field on the inactivation of E. coli O157:H7 within a given temperature range.